E. coli BW25113 ompA mutant vs wild type biofilm and E. coli BW25113 ompA/pCA24N_ompA vs ompA/pCA24N suspension cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Qun Ma
PROVIDER: E-GEOD-14064 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
OmpA influences Escherichia coli biofilm formation by repressing cellulose production through the CpxRA two-component system.
Environmental microbiology 20090706 10
Previously we discovered that OmpA of Escherichia coli increases biofilm formation on polystyrene surfaces (González Barrios et al., Biotechnol Bioeng, 93:188-200, 2006a). Here we show OmpA influences biofilm formation differently on hydrophobic and hydrophilic surfaces since it represses cellulose production which is hydrophilic. OmpA increased biofilm formation on polystyrene, polypropylene, and polyvinyl surfaces while it decreased biofilm formation on glass surfaces. Sand column assays corro ...[more]
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Project description:The global transcriptional regulator Hha of Escherichia coli controls hemolysin activity, biofilm formation, and virulence expressions. Earlier, we have reported that Hha represses initial biofilm formation and disperses biofilms as well as controls prophage excision in E. coli. Since biofilm dispersal is a promising area to control biofilms, here we rewired Hha to control biofilm dispersal and formation. The Hha variant Hha13D6 was obtained to have enhanced biofilm dispersal activity along with increased toxicity compared to wild-type Hha (Hha13D6 induces dispersal 60%, whereas wild-type Hha induces dispersal at early biofilms but not at mature biofilms). Toxic Hha13D6 caused cell death probably by the activation of proteases HslUV, Lon, and PrlC, and deletion of protease gene hslV with overproducing Hh13D6 repressed biofilm dispersal, indicating Hha13D6 induces biofilm dispersal through the activity of protease HslV. Furthermore, another Hha variant Hha24E9 was also obtained to decrease biofilm formation 4-fold compared to wild-type Hha by regulation of gadW, glpT, and phnF. However, the dispersal variant Hha13D6 did not decrease biofilm formation, while the biofilm variant Hha24E9 did not induce biofilm dispersal. Hence, Hha may have evolved two ways in response to environmental factors to control biofilm dispersal and formation, but both controlling mechanisms come from different regulatory systems. For the whole-transcriptome study of BW25113 hha/pCA24N-hha13D6 versus BW25113 hha/pCA24N-hha biofilm dispersal, cells were grown in 250 mL of LB glucose (0.2%) for 16 h at 125 rpm with 10 g of glass wool (Corning Glass Works, Corning, NY, USA) in 1 L Erlenmeyer flasks to form a robust biofilm (Ren et al., 2004) and incubated an additional 1 h with 1 mM IPTG to induce wild-type Hha and Hha13D6. Similarly, for the whole transcriptome study of BW25113 hha/pCA24N-hha24E9 versus BW25113 hha/pCA24N-hha biofilm formation, cells were grown in 250 mL of LB glucose (0.2%) containing 1 mM IPTG for 7 h at 250 rpm with 10 g of glass wool to form biofilms. Biofilm cells were obtained by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0°C, and RNALater buffer® (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from biofilm cells using a bead beater (Biospec, Bartlesville, OK, USA). cDNA synthesis, fragmentation, and hybridizations to the E. coli GeneChip Genome 2.0 array (Affymetrix, Santa Clara, CA, USA; P/N 511302). Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 2.0-fold (induced and repressed) cutoff for Hha13D6 DNA microarrays (standard deviation 1.3-fold) and 10.0-fold (induced) or 4.0-fold (repressed) for Hha24E9 DNA microarrays (standard deviation 4.0-fold), and if the p-value for comparing two chips was less than 0.05.
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2012-08-30 | E-GEOD-36779 | biostudies-arrayexpress
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2009-12-19 | E-GEOD-13902 | biostudies-arrayexpress
Project description:E. coli K-12 BW25113 mutant strain yncC expression in biofilm cells relative to E. coli wild-type strain expression in biofilm cells. All samples were cultured in LB with glasswool at 37C for 15 hours and E. coli K-12 MG1655 mutant yncC colony cells vs wild type colony cells in LB plates 15h 37C. Quorum-sensing signal autoinducer 2 (AI-2) stimulates Escherichia coli biofilm formation through the motility regulator MqsR that induces expression of the putative transcription factor encoded by yncC. Here we show YncC increases biofilm formation by decreasing mucoidy (corroborated by decreased exopolysaccharide production and increased sensitivity to bacteriophage P1 infection). Differential gene expression and gel shift assays demonstrated that YncC is a repressor of the predicted periplasmic protein-encoding gene ybiM which was corroborated by the isogenic yncC ybiM double mutation which repressed the yncC phenotypes (biofilm formation, mucoidy, and bacteriophage resistance). Through nickel-enrichment microarrays and additional gel shift assays, we found that the putative transcription factor B3023 (directly upstream of mqsR) binds the yncC promoter. Overexpressing MqsR, AI-2 import regulators LsrR/LsrK, and AI-2 exporter TqsA induced yncC transcription whereas the AI-2 synthase LuxS and B3023 repressed yncC. MqsR has a toxic effect on E. coli bacterial growth which is partially reduced by the b3023 mutation. Therefore, AI-2 quorum-sensing control of biofilm formation is mediated through regulator MqsR that induces expression of the transcription factor YncC which serves to inhibit the expression of periplasmic YbiM; this inhibition of YbiM prevents it from overexpressing exopolysaccharide (causing mucoidy) and prevents YbiM from inhibiting biofilm formation. Experiment Overall Design: Strains: E. coli K-12 BW25113 wild-type, mutant yncC Experiment Overall Design: and E. coli K-12 MG1655 wild-type, mutant yncC Experiment Overall Design: Medium: LB Experiment Overall Design: Biofilm grown on glasswool or colony cells growth in LB plates Experiment Overall Design: Time: 15 hours Experiment Overall Design: Temperature: 37C Experiment Overall Design: Cell type: biofilm or colony Experiment Overall Design: For the biofilm arrays in BW25113 background: Experiment Overall Design: Overnight cultures (16 h, 2.5 mL) of wild type E. coli BW25113 and BW25113 yncC in LB and LB with kanamycin (50 μg/mL), respectively, were used to inoculate 250 mL LB with 10 g of glass wool (Corning Glass Works, Corning, NY) for forming biofilm. After incubating at 37°C for 15 h with shaking (250 rpm), biofilm cells were prepared by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0°C as described before. The total RNA was isolated from biofilm cells as described previously. Experiment Overall Design: The E. coli Genechip antisense genome array (P/N 900381, Affymetrix, Santa Clara, CA) containing probes for more than 4200 open reading frames (ORFs) was used to analyze the complete E. coli transcriptome as described previously. Hybridizations were performed for 16 h and the total cell intensity was scaled automatically in the software to an average value of 630. The Gene Expression Technical Manual (Affymetrix) was followed for the procedures of DNA microarrays, and the GeneChip operating software (Affymetrix) was applied to analyze data of DNA microarrays. The data quality was assessed following the manufacturer's guidelines (GeneChip Expression Analysis: Data Analysis Fundamentals; Affymetrix) and also was based on the expected signals of E. coli BW25113 and the yncC mutant genotypes (e.g., signals of the deleted genes, araA and rhaA, were low for both BW25113 and BW25113 yncC, while the signal of yncC was low for the yncC mutant). A gene was identified as differentially-expressed when the P value based on the False Discovery Rate Method was less than 0.05 and the expression ratio was greater than threefold since the standard deviation for the expression ratio for all of genes was 2. Experiment Overall Design: for the colony arrays in MG1655 background: Experiment Overall Design: For the agar biofilm, fresh single colonies of wild-type E. coli MG1655 and MG1655 yncC were re-streaked on LB agar plates, incubated, and about 0.05 g of the colony cultures on LB plate were quickly transferred to 2-mL collection tubes. 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2008-04-07 | E-GEOD-8706 | biostudies-arrayexpress
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2012-04-02 | E-GEOD-34028 | biostudies-arrayexpress